Minor limonoid constituents from Swietenia macrophylla by simultaneous isolation using supercritical fluid chromatography and their biological activities

This study reports simultaneous isolation of three new limonoids (1–3), six known regio isomers (6, 7, 9–12), and three more known limonoids (4, 5, 8) from Swietenia macrophylla (S. macrophylla) seeds. Structures of these compounds were determined via extensive study of their 1D/2D-NMR and mass spectral data. Known limonoids (4–12) were identified by comparing their physical and spectroscopic data with literature values. A novel environmentally friendly supercritical fluid chromatography (SFC) technique facilitated simultaneous and rapid separation of these compounds. The pharmacological activities of the new limonoids were investigated.


Introduction
Mahogany, or Swietenia macrophylla (S. macrophylla), is a signicant medicinal plant cultivated in tropical and subtropical regions.Traditional medicine employs all parts of the S. macrophylla to cure a range of human ailments. 1The study of the phytochemical components found in S. macrophylla's various plant sections demonstrated the plant's abundance in triterpenes, referred to as limonoids, and their derivatives. 2These limonoids are prevalent in citrus fruits of S. macrophylla with a bitter taste and sweet or sour scent.With a furan ring attached at the C-17 position, they usually exhibit high degrees of oxidation and skeletal rearrangements.These carbon framework containing limonoids include andirobin, gedunin, mexicanolide, phragmalin, and D-ring-opened phragmalin (Fig. 1).2b Puried limonoids from S. macrophylla have been employed in several medical treatments such as hypertension, 3 anti-diabetic, 4,5 anti-bacterial, 6 anti-inammatory, antimicrobial, anti-malarial, anti-oxidant, anti-tumor, treatment of dengue virus [7][8][9][10] and hypolipidemic activity. 11,12he primary botanical families known to contain limonoids are Meliaceae, Rutaceae, and Simaroubaceae.Within these plant families, various species have been identied as rich sources of limonoids.These compounds are oen found in the seeds, fruits, leaves, and bark of such plant species.More than 300 different limonoids have been identied from numerous plant sources, 13,14 and there are undoubtedly many more limonoids yet to be found.
Medicinal plants possess complex matrices that allow them to produce compounds showing wide variety of biological activities.Purication, measurement, separation, and characterization of bioactive compounds from extracts of plant material have never been easy.Fortunately, several innovative techniques have emerged that provide compelling evidence for improving the method's sensitivity, selectivity, and run times in the evaluation of therapeutic herbs.Therefore, selecting a simple and appropriate separation technique for natural product separation has become increasingly crucial in recent years.
The development of new, supercritical technology instruments for a wide variety of column chemistries and uid chromatography, along with innate technological qualities, have made supercritical uid chromatography (SFC) a stand-in and renowned analytical platform for research on therapeutic plants.The SFC is a green technique, offers several advantages over Prep-HPLC such as easy to use, faster separations, and availability of wide variety of SFC stationary phases with diverging properties.Furthermore, SFC can assess substances that are insoluble in Prep-HPLC solvents, leading to more precise and well-resolved peaks.SFC is a desirable technique for the separation of non-polar natural components such as terpenes, fatty acids, vitamins, sterols, and also moderate-tostrong polar components using modiers. 15,16This technique is particularly well-liked for the enantio separation of chiral compounds. 17Supercritical uids have higher diffusivities and lower viscosities, which make them more efficient, easier to scale up, and need less time to analyse.It makes drying at lower temperatures easier and maintains the stability of the phytochemical elements because of the non-organic solvent systems. 18This perspective saw SFC as a promising substitute that safeguards the thermally labile compounds and gives a complimentary chromatography environment.3][34] However, only a few instances SFC purication processes were used to isolate plant extracts such as the ergostane triterpenoids from Antrodia camphorata, 35 polyphenols from Mangifera indica Linn, 36 carsonic acid from rosemary extracts, 37 and Piper kadsura. 381][42][43] Isolating pure components was laborious, and many compounds degraded during drying.Due to the complexity and abundance, traditional isolation of limonoids from S. macrophylla posed challenges, due to their low UV absorbance (steroidal skeleton) and similarity in adsorption behavior. 22Availability of no report on the use of SFC for the study of phytochemicals from S. macrophylla set out the goal of the current work to use the SFC approach to separate the limonoids from the acetone and ethanol extracts of S. macrophylla seeds.

Results and discussion
Harvested fruits of S. macrophylla were collected from Salem, Tamilnadu, India, in the northern hemisphere of Asia, which is situated at latitude 11.65376 and longitude 78.15538.Aer shade drying and grinding the seeds to coarse powder, it was repeatedly defated using hexane at room temperature (Fig. 2) and successively extracted with chloroform, acetone, ethanol, methanol, and water.
Even though each extract had a variety of phytoconstituents, the acetone and ethanol extracts which provided higher quantity of crude phytoconstituents were selected for further study.Steps were taken to develop an effective, simultaneous, one-step SFC purication of several phytochemicals.To optimise SFC conditions, several factors encompassing the stationary phase, mobile phase, and other instrumental parameters were considered.Three achiral columns namely, Princeton 2-ethylpyridine, YMC Diol, and Daicel-P4VP, as well as nine chiral columns namely, LUX-i-Amylose-3, Chiralpak AD-H, LUX-Amylose-2, Chiralpak IG, Chiralpak AS-H, Chiral ART Amylose SA, Chiralpak IH, (R,R) Whelk-O1 and Chiralpak-IE, were examined.Due to the varying stereo-congurations of epimers, chiral columns typically exhibited signicantly higher resolutions compared to achiral columns.Stationary phases primarily composed of polysaccharides offer enhanced resolution in separating regio-, E/Z and non-enantiomeric isomeric mixtures of compounds. 44,45Among these polysaccharide phases, the Amylose-based Chiralpak-IE column, specically amylose tris(3,5-dichlorophenylcarbamate), was identied as providing superior separation of individual limonoid compounds while maintaining reasonable retention times (Fig. 3).Aer testing a variety of co-solvents, including ethanol, acetonitrile, isopropyl alcohol, the most effective co-solvent was identied as methanol and used along with supercritical carbon dioxide liquid as mobile phase for separating twelve different compounds from acetone and ethanol extracts.Structures of all compounds were determined by the interpretation of their 1D/ 2D-NMR, and additional spectroscopic data (Fig. 4).

Biological activities
S. macrophylla exhibits a wide range of pharmacological bene-ts, including antibacterial, anti-inammatory, antioxidant, antimutagenic, anticancer, antitumor, antidiabetic, antinociceptive, hypolipidemic, antidiarrheal, anti-infective, antiviral, antimalarial, acaricidal, and heavy metal phytoremediation activities. 58We examined the biological activities of newly discovered compounds 1, 2, and 3, in line with previously isolated compounds from S. macrophylla.Since several limonoids from S. macrophylla were the subject of thorough theoretical computations and computational analyses, 59,60 we did not conduct experimental tests or empirical validation to support these theoretical conclusions.
2.2.1.Anti-fungal activity.Triterpenoids, a signicant class of constitutive denes phytochemicals found in sufficient concentrations in S. macrophylla, are the true cause of the plant's increased ability to combat pathogenic fungus.Antifungal activity of newly isolated compounds 1, 2, and 3 were evaluated against Aspergillus Niger using the disc diffusion method, on Sabouraud dextrose agar plates and inoculated them with the fungal culture (Table 3).
Nevertheless, against Aspergillus Niger, none of the test compounds 1, 2 and 3 demonstrated any inhibition at 25 ml (0.05 mg), 50 ml (0.1 mg), 75 ml (0.15 mg), or 100 ml (0.2 mg). 61his information suggests that while these compounds may exhibit antibacterial activity while they do not possess the any efficacy against Aspergillus Niger, highlighting the importance of considering the target organism when assessing the effectiveness of antimicrobial agents.
2.2.2.Anti-inammatory activity.The anti-inammatory assay examines a substance's potential to prevent protein (1% bovine albumin) denaturation, a process linked to inammatory diseases like arthritis.Denatured proteins can result from tissue damage, exacerbating inammation.Percentage inhibition of denaturation is calculated using absorption at 660 nm.][64] The anti-inammatory effects of the newly isolated compounds 1, 2 and 3 (Table 4) revealed that while compounds 2 and 3 showed signicantly higher efficacy in causing protein denaturation, compound 1 performed poorly.It was also evident that compound 3's ability to denaturize proteins rises in direct proportion to concentration.
Compound 2 exhibited increased hemolytic activity with increase compound concentration, while compound 3 exhibited hemolytic activity, compound 1 exhibited no activity at all.However, the same compounds studied against heat-induced hemolytic activity all the compounds exhibited increased hemolytic activity with increasing concentration. 65,66.2.3.a-Amylase inhibition activity.In order to check hypoglycemic activity, the a-amylase inhibition activity was studied.67 Starch was used as a substrate, generation blue color Fig. 5 HMBC correlations for new limonoids 1, 2, and 3.
due to the presence of undigested starch was used as an indicator of enzyme inhibition (Table 5).Acarbose served as the positive control.Pre-incubation involved mixing 40 ml of substrate solution with acarbose or compounds 1, 2 and 3 at various concentrations (10-640 mg ml −1 ) before the addition of a-amylase (3 mg ml −1 ).Aer incubation at 37 °C for 15 minutes, the reaction was terminated with hydrochloric acid, followed by the addition of iodine reagent.Absorbance was then measured at 630 nm. 68,69tudy of alpha-amylase inhibitory potency revealed that compounds 1, 2 and 3 possessed potential hypoglycemic activity.As the concentration of compounds 1, 2 and 3 rises, there is a corresponding increase in the percentage inhibition of alpha-amylase.This suggests that higher concentrations of the compounds lead to greater inhibition of the enzyme's activity. 70,71Inhibiting its activity can be desirable for various reasons, such as controlling blood sugar levels in diabetes management or preventing spoilage in food processing.
2.2.4.Antimicrobial activity of the samples using the well diffusion method.The antibacterial activities of compounds 1, 2 and 3 were evaluated against Escherichia coli (MTCC 2412), Bacillus cereus (MTCC 2128), Staphylococcus aureus, and Klebsiella pneumoniae (MTCC 2451) using the agar-well diffusion method (Table 6).Ciprooxacin at 1 mg ml −1 was used as the positive reference.Mueller Hinton Agar (MH) plates were sterilized, inoculated with bacterial strains, and then incubated at 37 °C for 24 hours.The diameter of inhibition zones (DIZ) around the wells was measured in millimetres to assess antibacterial activity.Three replicates of each experiment were performed.
The antibacterial activity of the compounds 1, 2 and 3 increases with the increase in concentration.This means there are more molecules available to target and inhibit the growth or kill the bacteria.As a result, the antibacterial effect becomes stronger because there's a higher likelihood of these compounds effectively neutralizing or eliminating the bacteria they encounter.
2.2.5.Anti-mutagenic activity by comet assay.The comet assay, or single-cell gel electrophoresis (SCGE), is a method used to quantify DNA damage in individual cells.This is a conventional method used to assess DNA damage and repair, monitor biological samples, and conduct tests to determine the potential for genetic damage. 71ompound 1 exhibited notable genotoxic effects on human lymphocytes (Table 7).This suggests that when exposed to compound 1, there was an observable damage to the genetic material of the lymphocytes.Such damage can have serious implications, including increased risk of cancer or other genetic disorders.On the other hand, compounds 2 and 3 show no discernible impacts on human lymphocytes in terms of genotoxicity.This implies that exposure to compounds 2 and 3 does not result in observable genetic damage or mutations in the lymphocytes.

General experimental procedures
Using a digital polarimeter (JASCO P-2000), optical rotations were measured.Using a Shimadzu 2000 FT-IR spectrophotometer, IR spectra were acquired.NMR spectra were acquired using a 400 MHz Bruker Avance.A Waters ACQUITY UPLC H-Class coupled with SQ Detector-2 mass spectrometer was used to gather ESIMS data.ESIMS data with high resolution were acquired using an exploris 240 Thermo orbit trap.SFC purications using Waters SFC-150 mgm equipment and SFC analysis performed on Waters SFC-Investigator instruments.Chiralpak-IE (4.6 × 250 mm; 5m id) column from Diacel chiral

Extraction and isolation
Aer being processed using an electronic grinder for a week to a coarse powder, the seeds were weighed, shade-dried, and stored in a dry place.500 g of dry powder were continuously cold extracted using hexane residue, followed by chloroform, acetone, ethanol, methanol, and water three times each.A rotary evaporator was utilized to eliminate the solvents from every extract.The extracts were then kept at −70 °C for 48 h, and a freeze-dryer (Labconco Corporation, Denmark) was used to freeze-dry them under a vacuum for 24 h at −40 °C.Strictly sealed glass bottles containing each dried extract were kept at 4 °C.The extraction yield from S. macrophylla seeds was found to be highest in ethanol solvent (68 g with 13.6%) and lowest in aqueous solvent (28.0 g with 5.6%), according to a quantitative evaluation of the extracts observed from the seeds using different solvents.

Optimizing SFC conditions for efficient phytochemical separation
We focused on developing an effective one-step SFC method for isolating phytochemicals, prioritizing compounds separable by acetone and ethanol.SFC offers superior separation efficiency and additional benets such as online coupled processes, faster separations, reduced solvent consumption, wide applicability, and easy analyte recovery.SFC conditions were optimized, including stationary phase, mobile phase, and instrumental parameters, for separating limonoids from S. macrophylla seed extracts.Chiral columns showed higher resolution than achiral ones due to the presence of various positional isomers.
The Chiralpak-IE column was identied as optimal for limonoid separation using methanol as the most effective solvent.Key parameters included a column temperature of 35  °C, a 30% co-solvent composition, a sample volume of 10 ml, a ow rate of 4.0 ml min −1 , and 100 bars of back pressure.These optimized conditions were applied in the analytical SFC for isolating compounds from ethanol and acetone extracts.Seven compounds 1, 2, 4, 5, 6, 7, and 8 were isolated from acetone extract, and ve compounds 3, 9, 10, 11, and 12 from ethanol extract, even with extremely low abundances (2-3%), demonstrating the efficiency of the SFC approach.
The SFC purication was not performed for extracts of hexane, chloroform, methanol, and water due to their low solubility, poor peak shape, and retention behaviour.The purity of isolated compounds was conrmed using SFC investigation, and certain compounds were disregarded for further research as they were well-known and extensively reported in the literature.Initial LCMS analysis conrmed the presence of the same compounds in multiple extracts, leading to the selection of ethanol and acetone extracts for SFC purication.

Conclusion
In conclusion, we demonstrated for the rst time that SFC could be used as a powerful technique for the simultaneous and superior separation of twelve compounds, in particular  a Disc diffusion method (radius of the zone of inhibition mm).
limonoids from S. macrophylla in a single-step.An optimum condition was arrived at based the study of different parameters such as column, pressure, temperature, and co-solvent composition.Compounds 1, 2, and 3 were previously unknown and challenging to isolate by conventional natural products separation methods.This study further shows that using the SFC isolation technique, even trace-level (2-3%) phyto constituents such as limonoids 1, 2, 3, 9, 10, 11 and 12 could be conveniently separated from complex mixtures.The use of recyclable carbon dioxide as the primary solvent, reduced use of organic solvent, efficient and simultaneous separation of minor natural product constituents by means SFC makes our separation technique sustainable, green and eco-friendly.These results are unattainable through traditional chromatographic methods, efficient alternative, and are unprecedented in the literature on S. macrophylla.Further, the new limonoids 1, 2 and 3 assessed for antimicrobial, anti-inammatory, hemolytic, and genotoxic properties disclose novel biological activities.Compounds 1, 2 and 3 shows good hypoglycemic activity useful in controlling blood sugar levels and diabetes management, signicant antibacterial activity and no anti-fungal activity.Compounds 2 and 3 showed signicant anti-inammatory activity and anti-mutagenic activity.Whereas compound 1 showed poor anti-inammatory activity and genotoxic effects on human lymphocytes.Studying mechanism and therapeutic effects of the limonoids isolated from S. macrophylla, could facilitate the discovery of potential new drugs.

Fig. 2
Fig. 2 Room temperature extraction of S. macrophylla seeds using different solvents.

Fig. 4
Fig. 4 Molecular structure of new and known limonoids isolated from the seeds of Swietenia macrophylla.

Table 2
Key HMBC correlations of new compounds 1, 2 and 3

Table 3
Anti-fungal activity of selected test samples by disc diffusion method a a NIno inhibition.

Table 5
Alpha amylase % inhibition activity of compounds 1, 2 and 3 a % inhibition= (absorbance-control − absorbance-test)absorbance-control ×100, where A-control = absorbance of the blank control and A-test = absorbance of the test sample. a

Table 7
Genotoxic activity of the compounds on human lymphocytes © 2024 The Author(s).Published by the Royal Society of Chemistry RSC Adv., 2024, 14, 26637-26647 | 26645 Paper RSC Advances